Methods using polarimetry, colorimetry, reductimetry and a variety of chromatographies have been developed as methods for quantitative analysis of sugars such as sucrose and glucose. These methods, however, are all poorly specific to sugars and hence have poor accuracy. Among these methods, the polarimetry is simple in manipulation, but is largely affected by the temperature during the manipulation. Therefore, the polarimetry is not suitable as a method for simple quantification of sugars at home, etc. for ordinary people.
Recently, various types of biosensors utilizing the specific catalytic action of enzymes have been under development.
The following will describe a method of glucose quantification as one example of the method of quantifying a substrate contained in a sample. As an electrochemical method of glucose quantification, a method using glucose oxidase (EC 1.1.3.4: hereinafter abbreviated to GOD) and an oxygen electrode or a hydrogen peroxide electrode is generally well-known (see “Biosensor” ed. by Shuichi Suzuki, Kodansha, for example).
GOD selectively oxidizes β-D-glucose as a substrate to D-glucono-δ-lactone using oxygen as an electron mediator. In the presence of oxygen, oxygen is reduced to hydrogen peroxide during the oxidation reaction process by GOD. The decreased amount of oxygen is measured by the oxygen electrode, or the increased amount of hydrogen peroxide is measured by the hydrogen peroxide electrode. Since the decreased amount of oxygen and the increased amount of hydrogen peroxide are proportional to the content of glucose in the sample, glucose quantification is possible based on the decreased amount of oxygen or the increased amount of hydrogen peroxide.
The above method utilizes the specificity of the enzyme reaction to enable accurate quantification of glucose in the sample. However, as speculated from the reaction process, there is a drawback that the measurement results are largely affected by the oxygen concentration of the sample, and if the oxygen is absent in the sample, the measurement is infeasible.
Under such circumstances, glucose sensors of new type have been developed which use as the electron mediator potassium ferricyanide, an organic compound or a metal complex such as a ferrocene derivative and a quinone derivative without using oxygen as the electron mediator. In the sensors of this type, reduced form electron mediator resulting from the enzyme reaction is oxidized on a working electrode, and the concentration of glucose contained in the sample can be determined based on the amount of this oxidation current. At this time, on a counter electrode, a reaction in which oxidized form electron mediator is reduced to produce reduced form electron mediator proceeds. With the use of such an organic compound or metal complex as the electron mediator in place of oxygen, it is possible to form a reagent layer while a known amount of GOD and the electron mediator are carried in a stable state and a precise manner on the electrode, so that accurate quantification of glucose is possible without being affected by the oxygen concentration of the sample. In this case, it is also possible to integrate the reagent layer, in an almost dry state, with an electrode system, and hence disposable glucose sensors based on this technique have recently been receiving a lot of attention. A typical example thereof is a biosensor disclosed in Japanese Patent Publication No. 2517153. In such a disposable glucose sensor, it is possible to measure glucose concentration easily with a measurement device by simply introducing a sample into the sensor connected detachably to the measurement device. The application of such a technique is not limited to quantification of glucose and may be extended to quantification of any other substrate contained in the sample.
In the above-mentioned conventional biosensors, even after introduction of the sample into the biosensor, part of the sample is exposed to outside, and part of the sample therefore evaporates during the measurement, possibly causing a change in substrate concentration of the sample. Although the conventional biosensors are capable of measuring a sample in the order of microliter, development of biosensors capable of measuring a sample in a further smaller amount has been anxiously hoped for from various fields in recent years. However, with the reduction of the sample, even a slight change in substrate concentration may cause variations in measured values in the conventional biosensors.
Therefore, an object of the present invention is to provide an analytical element which can prevent evaporation of a sample during measurement and therefore quantify a substrate using a very small amount of sample with high accuracy and which can prevent scattering of the sample during and after the measurement and is therefore hygienically excellent.
Another object of the present invention is to provide a measuring device comprising such an analytical element and a method of substrate quantification.